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For 200 years—since 1817—the Academy has brought together extraordinary people working at the frontiers of discovery. Among the oldest scientific organizations in the United States, it has become not only an enduring cultural institution in New York, but also one of the most significant organizations in the global scientific community.

Progressive Multifocal Leukoencephalopathy (PML) is, in the general population, a rare but serious demyelinating disease of the brain, often resulting in severe disability or death and is caused by infection of oligodendrocytes by the JC polyomavirus (JCV). It is believed that fifty to sixty percent of the population is sero-positive for JCV, indicating exposure to the virus. Despite the high prevalence of JCV infection in the human population, which typically leads to a chronic, asymptomatic infection, incidence of PML in the general population is very low. The pathogenesis of PML is not well understood. It is believed that the development of PML, while dependent on the presence of JCV, is the result of a confluence of viral and host risk factors, which may include an altered or compromised immune system such as in individuals with HIV-1 infection/acquired immune deficiency syndrome (AIDS), or patients undergoing chronic immunosuppressive therapies after organ transplantation, for treatment of lymphomas, or for autoimmune conditions, such as MS.

This 2-day conference will bring together basic science researchers, clinicians, physicians, epidemiologists, and regulatory experts from academia, industry, and government to address novel issues, current challenges, and future directions of basic and clinical research relevant to the mitigation, potential future cure, and risk stratification of PML. Topics of discussion will include JCV virology, PML pathogenesis, risk stratification and disease diagnosis and management. The meeting will feature a series of plenary lectures, short talk presentations selected from submitted abstracts, a poster session, and a closing panel discussion.

Conference Planning Committee

Claudio Carini, MD

Pfizer Inc.

Teresa Compton, PhD

Biogen Idec (Conference Chair)

Marion Kasaian, PhD

Pfizer Inc.

Theodora Salcedo, PhD

Bristol-Myers Squibb Company

Anne Vogt, PhD

F. Hoffmann-La Roche Ltd

Scientific Advisory Board

Leonard H. Calabrese, DO

Cleveland Clinic

Paola Cinque, MD, PhD

San Raffaele Scientific Institute

David B. Clifford, MD

Washington University School of Medicine

Robert L. Garcea, MD

University of Colorado at Boulder

Eugene O. Major, PhD

National Institute of Neurological Disorders and Stroke

Kenneth Tyler, MD

University of Colorado School of Medicine

Thomas Weber, MD

University of Hamburg

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* Presentation titles and times are subject to change.

Day 1: June 19, 2013

8:00 AM

Breakfast and Registration

8:45 AM

Welcoming Remarks Melinda Miller, PhD, The New York Academy of Sciences Teresa Compton, PhD, on behalf of the PML-Consortium

SESSION I: JCV Virology

JC polyomavirus (JCPyV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). The JCPyV life cycle begins with a multi-step attachment to virus receptors that facilitate the entry process. Following initial entry by clathrin dependent endocytosis, the virus is transported to the endoplasmic reticulum (ER) where host cell chaperones begin the process of viral uncoating. The genome along with some components of the capsid exit the ER and are targeted to the nucleus by unknown mechanisms. The process of retro-endosomal transport of the virus particle to the ER from the plasma membrane is reminiscent of the trafficking pathway used by the bacterial toxins ricin and shiga-like toxin (SLT). Recently, a chemical compound known as Retro-2 was shown to block retro-translocation of these toxins to the ER, and non-toxic doses of Retro-2 were demonstrated to protect mice from lethal challenge with ricin. We therefore asked whether this compound and related compounds that we developed would inhibit polyomavirus trafficking to the ER and subsequent infection. We demonstrate that Retro-2cycl and several derivatives potently inhibit infection by JCPyV, BK polyomavirus (BKPyV), and simian virus 40 (SV40). The compounds do not interfere with binding or uptake of virions from the plasma membrane, but instead inhibit retrograde trafficking to the ER, a step necessary for productive infection.

Leu/Ile/Phe-rich Domain of JC Virus Agnoprotein Plays Important Regulatory Roles in Dimer/Oligomer Formation, Protein Stability and Splicing of the Viral Transcripts Mahmut Safak, PhD, Temple University School of Medicine

Agnoprotein is a multifunctional regulatory protein of polyomaviruses, including JC virus (JCV). In the absence of its expression, JCV is unable to sustain its productive life cycle, which is evident from a poor replication behavior of the agnoprotein null mutants. It is a mainly a cytoplasmic protein with high concentrations accumulating around the perinuclear region of the infected cells, but, a small portion of the protein is also consistently detected in the nucleus, suggesting diverse functional roles for agnoprotein in the both compartments.

Understanding the molecular mechanisms underlying the functional roles of agnoprotein in JCV life cycle is still at infancy, in spite of much progress made in this respect. We have recently reported that agnoprotein forms stable dimer/oligomers mediated by a predicted an amphipathic α-helix, spanning amino acids (aa) 17 to 42. Deletion of the α-helix renders a replication incompetent virus. We have now further characterized this region by a systematic deletion and substitution mutagenesis and demonstrated that a Leu/Ile/Phe-rich domain (spanning aa 28-39) within the α-helix is critical for its structure and function. For instance, deletion of aa 30-37, severely affects the dimer/oligomer formation and stable expression of the protein. Mutagenesis data also revealed that in the absence of aa 34-36, the mutant virus exhibits defects in splicing of the viral transcripts, suggesting a novel regulatory role for agnoprotein in JCV gene regulation at the post-transcriptional level.

In addition, due to the basic amino acid composition and nucleocytoplasmic localization of agnoprotein, we reason that this protein may interact with JCV mRNA and be involved in export of the viral RNA from nucleus to cytoplasm. Indeed, our recent in vivo RNA-CHIP analysis revealed a specific interaction between agnoprotein and JCV RNA. Moreover, treatment of the infected cells with chromosomal region maintenance 1 (CRM1) inhibitor, Leptomycin B, resulted in a high level of accumulation of agnoprotein in the nucleus, suggesting involvement of agnoprotein in regulation of nucleocytoplasmic export of viral transcripts in a CRM1-dependent manner. Collectively, these findings demonstrate that the Leu/Ile/Phe-rich domain is critically important for the dimer/oligomer formation, stability and function of agnoprotein and thus represents a potential target for developing novel therapeutic agents for progressive multifocal leukoencephalopathy.

Most DNA viruses replicate in the nucleus, where viral capsid proteins and viral genomes spatially intersect to assemble infectious progeny. Electron microscopy of freeze-substituted, murine polyomavirus (MPyV)-infected mouse embryo fibroblasts identifies filamentous structures in the nucleus, adjacent to clusters of assembled virions. Tomographic reconstructions suggest that “bubble-like” structures travel the length of the filaments, and that virions are “shed” from their ends. We hypothesize that the filaments represent “virus factories,” where viral capsid proteins and viral genomes assemble to yield mature virions. Such a defined assembly structure would kinetically favor high fidelity virion production. These filamentous structures have been seen in brain lesions from humans with progressive multifocal leukocephalopathy.

To investigate the composition of these nuclear factories, we have analyzed the localization of viral and cellular proteins using confocal immunofluorescence microscopy, and replicating viral DNA using fluorescent in-situ hybridization (FISH). We have found that foci of replicating MPyV DNA co-localize with the viral T-antigen during early infection, and these foci expand as infection proceeds. Co-localizing to these sites of viral DNA we observed cell DNA repair proteins, such as Mre11 and Nbs1, and γH2A.x, along with activation of ATM kinase and CHK1. Inhibitors of ATM, Mre11, and Chk2 all reduce virus outputs. These observations support the view that polyomavirus replication utilizes the cellular DNA damage repair pathways for efficient viral DNA replication, as well as blocking the cell in cycle at the S to G2 stage, allowing continued access of viral replication to critical cellular factors.

JC Virus (JCV), the etiological agent of progressive multifocal leukoencephalopathy (PML), is a ubiquitous polyomavirus that causes significant morbidity and mortality in AIDS patients and people on certain immunosuppressive therapies. JCV lytically infects human glial cells, but can also nonproductively infect many cell types, including neurons. The transcriptional environment of the infected cell is the primary determinant of cellular tropism. Encapsidated JCV is nucleosomal, indicating that histone modifications may play a role in the JCV life cycle. Human neural multipotential progenitor cells were differentiated to progenitor derived astrocytes (PDAs) or progenitor-derived neurons (PDNs), infected with JCV, and analyzed by RT-PCR and chromatin immunoprecipitation 4 days after infection. T antigen RNA expression was 5-fold greater in PDAs than PDNs, and VP1 RNA expression was 7-fold greater in PDAs. Histone H3 levels on the regulatory region and coding regions of the JCV genome and acetylation of histone H3, which is a mark of active transcription, were similar in both cell types. Levels of heterochromatin protein 1g(HP1g), a mark of repressive chromatin, were higher on all regions of the viral genome in PDNs than PDAs. HP1gbinding correlated with binding to the JCV regulatory region of known JCV-repressive transcription factors, nuclear factor I-A, c-jun, and c-fos. Thus, it appears that negative regulators of transcription, including chromatin proteins and transcription factors, determine the reduced transcription of JCV in repressive cell types. Further analysis of determinants of transcription may yield insight into markers for PML susceptibility when determining treatments for patients requiring immunosuppressive therapies.

PML-Associated Mutations in the JC Polyomavirus Capsid Disrupt LSTc Binding Melissa S. Maginnis,PhD1 JC polyomavirus (JCPyV) is a ubiquitous human pathogen and the causative agent of the fatal, demyelinating disease Progressive Multifocal Leukoencephalopathy (PML). We have previously demonstrated that the JCPyV prototype strain Mad-1 binds specifically to alpha-2,6-linked lactoseries tetrasaccharide c (LSTc) to initiate infection of host cells. Specific residues in the viral capsid protein VP1 are responsible for mediating direct interactions with the a2,6-linked sialic acid of LSTc. Viral isolates from individuals with PML often contain mutations in the sialic acid-binding pocket of VP1 that are thought to arise from positive selection. We reconstituted these mutations in the Mad-1 strain of JCPyV and found that they were not capable of growth. The mutations were then introduced into recombinant VP1 and reconstituted as purified pentamer subunits in order to conduct binding studies and structural analyses. VP1 pentamers carrying PML-associated mutations were not capable of binding to permissive cells. High-resolution structural analysis revealed that these pentamers are properly folded but no longer bind to LSTc due to steric clashes in the sialic acid binding site. Reconstitution of the mutations into a JCPyV pseudovirus expression system revealed that pseudoviruses with PML-associated mutations were not infectious, nor were they able to engage sialic acid as measured by hemagglutination of human red blood cells. These results demonstrate that viruses from PML patients with single point mutations in VP1 disrupt binding to sialic acid motifs, suggesting that these viruses are either non-infectious or infect cells in a sialic acid-independent manner.

JC Virus Intranuclear Inclusions Associated with Promyelocytic Leukemia Nuclear Bodies Yukiko Shishido-Hara, MD, PhD1,2 Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder due to JC virus infection. Histopathologically, the viralinclusions are identified in the markedly enlarged nuclei of glial cells, as two distinctive patterns. The dot-shaped inclusions represent clusters of progeny virions accumulated at subnuclear domains called promyelocytic leukemia nuclear bodies (PML-NBs), while the full inclusions reflect the progenies dispersed to the entire part of the nucleoplasm. We analyzed JC virus capsid formation in use of a eukaryotic expression system providing virus-like particles (VLPs), and viral inclusions were further investigated in human brain tissues. We found that the major and minor capsid proteins cooperatively transport to the nucleus for capsid assembly at PML-NBs. In the presence of the agnogene, uniform VLPs were formed restrictedly at PML-NBs; while when the 5’ termini of the agnogene(nt 275-409) was deleted, pleomorphic VLPs were more randomly produced in the nucleus besides PML-NBs.The cells harboing VLPs were more severely degraded in the presence of agnogene than its absence. In the human brain tissues, the largest PML-NBs were shaped as spherical shells over 1 μm in diameter.The JC virus capsid proteins circumscribed the PML-NB surface, suggesting the active sites for progeny production.With advancement of demyelination, there appeared more full inclusions, in which PML-NB structures were disrupted. These results indicate that pathogenic JC virus progenies are efficiently re-produced at PML-NBs in the presence of agnogene. The PML-NBmay play important roles for JC viral progeny re-production, and also subsequence death of host glial cells.

Patient Keynote Address

A Patient and Family Perspective on PML Declan R. Walsh, Deferno Trust

In my presentation, I will share a patient and family perspective of the impact of a diagnosis of progressive multifocal leukoencephalopathy (PML). My wife, Dr. Natalie Murphy, was diagnosed with PML in 2009. At that time she was the first person to be diagnosed with Tysabri related PML in Ireland and Britain. I will provide the background, the key information about her illness and the events leading up to the diagnosis. Following on from that, I will share the impact that PML has had on her and her immediate family and friends, and her journey from diagnosis to where she is today.

As part of this journey, I set up the Deferno Trust on behalf of Natalie. It serves to provide information and family support to those affected by Tysabri related PML and provides a medium for those seeking information onimmune reconstitution inflammatory syndrome(IRIS)/PML from a non-medical point of view. I will share some of my experiences in meeting with others that were diagnosed with PML and give a brief summary of the nature of the comments and concerns of those that have been in touch through the website.

Finally, I would like to convey our views on what we, as patients and families, would require from all of the various elements of the medical industry and seek a collaborative approach to ensure that the patients and their families remain at the forefront of the efforts in finding a solution to this incurable disease.

SESSION II: Pathogenesis

Immunology of PML Roland Martin, MD, University Hospital Zurich, Universität Zürich Immune control of JC polyoma virus (JCV) involves all aspects of the adaptive immune system including JCV-specific B cells/antibodies, CD4+ and CD8+ T cells. In immunocompromised patients, progressive multifocal leukeoencephalopathy (PML) probably develops in a multi-step process including perturbed JCV “homing”, mutation to glio-/neurotropic JCV strains, and compromise of helper (CD4+) and effector T cells (CD8+), and also B cell/antibodies. We have recently characterized the T cell responses in the brain of PML and PML immune reconstitution inflammatory syndrome (PML-IRIS), and also of a first case of cerebellar granule neuronopathy (CGN), which occurred during treatment with natalizumab. Main findings in the brain infiltrate of PML-IRIS are the predominance of CD4+ T cells that are specific for JCV (VP1) epitopes and frequently express a Th1-2 phenotype, i.e., secrete large amounts of interferon-γ and interleukin-4. Most prominently expressed T cell clones in the brain tissue use T cell receptors that are capable of interacting with two or more different HLA/peptide complexes (cross-restricted). In the GCN case, the brain-infiltrating T cells involve clonally expanded CD4+ and CD8+ populations, and T cells that co-express CD8+ and CD4+ appear most important.

Based on our data we reasoned that boosting a protective CD4+ T cell response might be particularly important for improving overall JCV immune control in PML and that this would also aid the effector arms (CD8+ T cells and antibodies). The successful treatment of two PML patients with idiopathic CD4+ lymphopenia and secondary immunocompromise by a combination of recombinant human interleukin-7 (rh-IL-7) and vaccination with JCV VP1 protein and an adjuvant supports our hypothesis. Finally, in a parallel program to develop a passive PML vaccine, we have isolated high affinity human monoclonal antibodies against JCV virus-like particles from PML-IRIS patients. These data indicate that a better understanding of JCV immune control during physiologic conditions and in PML promises to lead to an effective treatment and prophylaxis.

The first evidence of polyomavirus (PyV) infections in humans was obtained in 1964 by the discovery of typical non-enveloped particles in progressive multifocal leukoencephalopathy (PML). The subsequent isolation by cell culture proved difficult, but was eventually reported as successful in 1971 when using primary human fetal glia cells and post mortem PML tissue from a patient J.C. whose initials the virus has carried since. Independently, the identification of another polyomavirus was reported in 1971, but from the urine of a kidney transplant patient and also named according to the patient’s initials, B.K.. Both JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) share several characteristics: high seroprevalence in the general population of 50% - 90%; persistence in the renourinary tract; asymptomatic shedding in urine of 5% - 35% of the seropositive individuals, and limited cross-protection and interference as revealed by T cell reactivity, but not by antibody activity. However, unlike JCPyV, BKPyV remained an “orphan” virus until it was associated with hemorrhagic cystitis in allogeneic hematopoietic stem cell transplant patients and with nephropathy in kidney transplant patients. The discovery of the sequence of BKPyV viruria, viremia and organ invasive allograft disease in kidney transplant patients provided a new paradigm of PyV pathogenesis, and offered new diagnostic tools for screening and monitoring patients at risk. Moreover, rapid viral turnover kinetics with a plasma half-life of <2 hours were derived from graft nephrectomy studies of patients with stable steady-state plasma viral loads. Importantly, the emergence of more virulent BKPyV variants bearing rearranged non-coding control regions, akin to JCPyV variants found in PML, could be documented in real time in individual patients and linked to higher viral loads and more advanced histopathology. The histological characterization of BKPyV nephropathy revealed three major patterns described as cytopathic-noninflammatory (A), cytopathic-inflammatory (B), and fibrosis-atrophic, which predict progressive organ failure. Interestingly, JCPyV nephropathy has also been diagnosed in renal transplant patients, but only rarely. Significant differences in the virus-host interaction have been noted between BKPyV and JCPyV in healthy humans and in kidney transplant patients with nephropathy, which are of interest for the current research and understanding of JCPyV-PML (Rinaldo & Hirsch,APMIS,in press).

JC virus (JCV) is the etiologic agent of several diseases of the central nervous system, including progressive multifocal leukoencephalopathy (PML), JCV granule cell neuronopathy (JCV GCN) and JCV encephalopathy (JCVE). Between 30-90% of healthy adults have antibodies against JCV, depending on the assay and population tested. However, the humoral immune response is not sufficient to prevent reactivation of the virus in the setting of immunosuppression, leading to the development of CNS diseases. The cellular immune response, mediated by CD4+ and CD8+ T cells, plays a crucial role in the containment of PML. The presence and function of JCV-specific T cells can be measured by chromium release assay, tetramer staining, Elispot and intracellular cytokine staining assays. Using these techniques, a constant observation is that the number of JCV-specific T cells in circulating blood is very low, which makes ex vivo measurement of their function challenging, resulting in the need for in vitro stimulation with JCV peptides or antigen to obtain robust responses.

Despite their low numbers, JCV-specific CD8+ cytotoxic T lymphocytes play a crucial role, and their presence has been associated with a favorable clinical outcome and improved survival. Conversely, T-cells have been implicated in the pathogenesis of the immune reconstitution inflammatory syndrome (IRIS). IRIS is a frequent manifestation in PML patients with AIDS upon starting antiretroviral medications, and occurs in most multiple sclerosis patients with natalizumab-associated PML, after discontinuation of the medication. Indeed, IRIS may lead to brain edema, herniation and death.

We will review the beneficial and potentially deleterious effects of the cellular immune response in PML patients with and without IRIS, discuss strategies and pitfalls in detecting JCV-specific T cells in blood and central nervous system, as well as implications for the clinical management of these patients.

SHORT TALKS SELECTION

Progressive multifocal leukoencephalopathy (PML) is caused by uncontrolled replication of JC virus (JCV) in the white matter of the brain. A great majority of adults, including patients at risk of PML, have serum antibodies capable of neutralizing lab-adapted JCV strains. It remains unclear why these antibodies fail to protect patients against PML. Intriguingly, recent reports indicate that JCV isolates from the cerebrospinal fluid (CSF) of PML patients often contain distinctive mutations in the viral major capsid protein VP1. This observation raises the possibility that JCV mutants found in the CSF of PML patients can evade recognition by neutralizing antibodies. To explore this idea, we developed a set of JCV reporter vectors (pseudoviruses) and performed neutralization serology. Our results show that, while most healthy individuals have serum antibodies capable of neutralizing a broad range of wildtype and PML-derived JCV mutants, a small percentage of tested sera fail to neutralize a subset of PML-variant strains. The results suggest that rare individuals have a “blind spot” in their JCV-neutralizing antibody repertoire, resulting in poor humoral protection against JCV mutants associated with PML. We speculate that such individuals might be at increased risk of developing PML under conditions of T cell immunosuppression. Our model suggests two potential clinical approaches. First, it might be possible to treat PML by administering a humanized monoclonal antibody capable of neutralizing a broad range of JCV variants. Second, administration of a virus-like particle vaccine based on the capsid protein VP1 might help broaden the neutralizing antibody repertoire in patients at risk of developing PML.

Development of a Mouse Model of Polyomavirus-Induced Central Nervous System Disease Elizabeth L. Frost, BS1,2

Mechanisms underlying progressive multifocal leukoencephalopathy (PML) pathogenesis, including whether capsid mutations alter tropism of JC virus (JCV) to increase replication in the CNS and whether inflammation contributes to neuropathology, are poorly understood due to lack of a tractable animal model. Using mouse polyomavirus (MPyV) infection in mice, we seek to develop an animal model of polyomavirus-induced CNS disease to elucidate the role that VP1 mutation and/or immunosuppression play in JCV-PML pathogenesis. Intracerebral (i.c.) injection of MPyV in immunocompetent C57BL/6 mice resulted in extensive demyelination during persistent infection. Introduction of a PML-associated single amino acid substitution in JCV (VP1) into the analogous position in MPyV did not result in increased virus levels in the brain, but showed decreased replication in peripheral organs. Flow cytometric analysis of CNS-derived lymphocytes showed a robust infiltration by MPyV-specific CD8+ T cells, but with minimal contraction of this population through 30 days post-infection. Interestingly, CD8+ T cell-deficient (i.e., CD8α-/-) mice showed increased virus levels in the brain but not in the spleen, indicating a dependence on CD8+ T cells for MPyV CNS immunosurveillance. In addition, during persistent CNS infection, a significant population of CD8+ T cells having a tissue-resident memory phenotype (CD103+CD69+CD62Llo) gradually developed in the brains of i.c. inoculated C57BL/6 mice. Ongoing flow cytometry studies utilizing an MPyV-specific T cell receptor (TCR)-transgenic system will determine whether administration of CD8-depleting antibodies and/or VLA-4 blockade during persistent infection affects cells already infiltrating the CNS or prevents replenishment of the CD8+ T cell pool by de novo recruitment from the periphery.

Progressive Multifocal Leukoencephalopathy (PML) is caused by reactivation of JC virus (JCV). There is no animal model of PML since JCV only infect humans. Humanized mice provide an ideal model for studying JCV immune response. Immunodeficient mice, NOD/SCID/IL-2R, were engrafted with human hematopoietic cells and thymus. JCV(Mad-4 or CY archetype) strains were injected intraperitoneally. Mice were sacrificed between 47-103 days after initial infection. All mice tolerated intraperitoneal injection and infection of JCV without developing any illness, similar to primary infection in humans. JCV DNA was detected in the plasma and whole blood of JCVMad-4 mice between day 14-67. Two JCVMad-4 mice also had detectable JCV DNA in the urine. Anti-JCV IgM was detected in 7/13 JCVMad-4 and 4/14 JCVCY infected mice between days 47 and 90 after infection. On post-infection day 108, 3/7 JCVMad-4 infected mice had detectable JCV specific T-cell response in both ICS and ELISPOT assays. The humanized mice provide an animal model of immune response to JCV. Primary infection is asymptomatic and virus is rarely detected in whole blood and plasma, but is occasionally excreted in the urine. JCVMad-4-infected mice can develop both humoral and cellular immune responses to JCV. This model may prove very valuable for studying JCV host immune response and determinants of viral latency and reactivation.

Development of a Nonhuman Primate Model of Polyomavirus Disease in Bolivian Squirrel Monkeys John A. Vanchiere, MD, PhD1

The lack of a nonhuman primate model of human polyomavirus disease has hindered the development of therapies directed at prevention and control of polyomavirus-associated renal and neurologic disease. The squirrel monkey polyomavirus (SMPyV) is commonly found in the stool, spleen and mesenteric lymph nodes of immunologically intact and immune suppressed Bolivian squirrel monkeys and, as such, may be useful for development of a nonhuman primate model of polyomavirus disease. We treated Bolivian squirrel monkeys with rituximab for 16 weeks and demonstrated profound depletion of CD20-positive lymphocytes, as measured by flow cytometry. Treatment with an anti-CD8 monoclonal antibody lead to transient depletion of CD8-positive lymphocytes. In combination, these treatments resulted in persistent excretion of the SMPyV in the urine of four of six treated animals and the production of SMPyV variants with rearrangements of the non-coding control regions in the urine of two of the four virus-excreting animals. High viral loads (10e3 -10e5 genomes/μg of DNA) of SMPyV were observed in kidney tissue in two of three treated animals from which tissue was available. Moderate to severe peritubular and juxtaglomerular CD3+ inflammatory cell infiltration of the kidneys was observed by immunohistochemistry. One animal developed multifocal encephalitis with granuloma formation and accumulation of glial cells and CD3+ inflammatory infiltrates within the brain parenchyma. The findings are consistent with an early-stage model of polyomavirus disease in both the kidney and brain. Long-term studies and the development of reagents for the study of SMPyV-specific immune responses are necessary to more fully characterize this model.

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